Specific Interaction of DNA Polymerase β and DNA Ligase I in a Multiprotein Base Excision Repair Complex from Bovine Testis

Abstract

To investigate potential interaction of ␤-pol with other BER protein(s), we developed affinity chromatography matrices by crosslinking purified rat ␤-pol or antibody against ␤-pol to solid supports. Crude nuclear extract from bovine testis was applied to these affinity columns, which were then extensively washed. Proteins that bound specifically to the affinity columns were co-eluted in a complex with ␤-pol. This complex had a molecular mass of approximately 180 kDa and was able to conduct the complete uracil-initiated BER reaction. The BER complex contained both ␤-pol and DNA ligase I. An antibody to ␤-pol was able to shift the complex in sucrose gradients to a much larger molecular mass (>300 kDa) that again contained both ␤-pol and DNA ligase I. Furthermore, DNA ligase I and ␤-pol were co-immunoprecipitated from the testis nuclear extract with anti ␤-pol IgG. Thus, we conclude that ␤-pol and DNA ligase I are components of a multiprotein complex that performs BER. Base excision repair (BER)1 is initiated by enzymatic removal of a damaged or inappropriate base residue in DNA by a DNA glycosylase. This class of enzymes recognizes and removes a variety of single base lesions in DNA (1). Uracil-DNA glycosylase (UDG), which catalyzes the removal of uracil from a G:U mismatch to initiate BER, is the most extensively studied of these enzymes (2-4). The apurinic/apyrimidinic (AP) site is generated by DNA glycosylase (5) and also by spontaneous cleavage of the glycosidic bond, in particular those involving purines (6). Both procaryotes and eucaryotes contain AP endonucleases (APE) that incise the phosphodiester backbone of DNA at the 5Ј side of an AP site leaving a 3Ј-OH and 5Ј-deoxyribose phosphate (5, 7). Subsequently, the 5Ј-deoxyribose phosphate is removed either by a deoxyribose phosphodiesterase (dRpase) or by a 5Ј33Ј exonuclease, generating a onenucleotide gap with 3Ј-OH and 5Ј-phosphate termini (8 -11). The single nucleotide gap is then filled by DNA polymerase I in Escherichia coli or by DNA polymerase ␤ (␤-pol) in mammalian cells (12)(13)(14). Finally, the nick is sealed by DNA ligase. Dianov and Lindahl (12) ␤-Pol is a 39-kDa monomeric enzyme that lacks intrinsic nuclease activity (18). However, it has recently been reported to possess 5Ј-deoxyribose phosphodiesterase activity (19). In recent years, ␤-pol has been implicated in the repair of several lesions that are repaired by the BER pathway, among them G:T and G:U mismatches (13,14,20), abasic sites (21-23), adducts introduced by monofunctional alkylating agents (14,24), and in vitro bypass of a d(GpG)-cisplatin adduct (25). ␤-Pol also appears to function in some specialized cases of DNA replication (26 -30).Mammalian ␤-pols have been cloned and overexpressed in E. coli (31-34). The recombinant proteins are fully active in DNA synthesis and have been exploited for structure-function studies (35-38). We recently generated monoclonal antibodies and several neutralizing polyclonal antibodies specific for mammalian ␤-pols (13, 39). Using these reagents, we h...

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